BRPI0707395A2 - use of a pharmaceutical composition containing epidermal growth factor microspheres - Google Patents

Abstract

USE OF A PHARMACEUTICAL COMPOSITION CONTAINING EPIDERMIC GROWTH FACTOR MICROSPHERES. A pharmaceutical composition containing epidermal growth factor loaded microspheres for parenteral administration to the lower limbs of diabetic patients afflicted with chronic ischemic skin lesions. The composition of the present invention, in contrast to the prior art, is useful because it reduces the frequency of administration during treatment and allows wound healing to be faster compared to the administration of equivalent amounts of free factor in solution.

Description

"USE OF A PHARMACEUTICAL COMPOSITION CONTAINING EPIDERMIC GROWTH FACTOR SPHERES"

Field of technique

The present invention relates to a pharmaceutical composition containing microspheres encapsulated Epidermal Growth Factor for parenteral administration to the lower limbs of diabetic patients affected by chronic ischemic skin lesions in order to prevent amputation of said limbs.

Prior Art

Diabetes mellitus is the main non-traumatic risk factor for lower limb amputation. Foot ulceration is a significant complication of diabetes with an annual incidence slightly greater than 2% (Abbott CA, et al (2002). The North-West Diabetes Foot CareStudy: incidence and risk factors for new-onset diabetic foot ulceration) patient cohort Diabet Med 19 (5): 377-84). It is estimated that 15% of diabetes patients will develop ulcers at some point in their lives (Reiber GE (1996) The Epidemiology of Diabetic Foot Problems. Diabet. Med. 13 Suppl 1: S6-11) and that around 10% -30 % of those with ulcers will progress to amputation (Lipsky BA (2004) Medical treatment of diabetic foot infections. Clin. Infect. Dis. 39 Suppl2: S104-14). The 5-year mortality of patients with lower limb amputation is 50-60% (Reiber G. E. (1996) The Epidemiology of Diabetic Foot Problems. Diabet. Med. 13 Suppl 1: S6-11).

Several methods have been used to treat the patient with diabetic foot which include strict metabolic control, prophylaxis of modifiable risk factors, debridement, dressing, antimicrobial treatment of infections, elimination of pressure from the injured area, use of skin grafts, growth factors. and the use of derascularization methods in case of indication. After strict metabolic control, debridement is the most important step for the healing of diabetic ulcers and it is necessary to perform it before any other local treatment modality. This is the removal of all non-viable and infected tissue (including bones) from the injured region as well as the surrounding callus tissue.

The use of patches in diabetic foot ulcers is common and although several types of patches have been studied, the superiority of one over the other is unknown. In addition, there have been few studies and even directed to low-grade ulcers requiring further evidence from clinical trials to demonstrate the effectiveness of these methods. Among the new types of patches studied in controlled clinical trials are patches based on semipermeable polymeric membrane, promogran (collagen matrix), alginate, carboxymethylcellulose, hyaluronan and subatmospheric pressure (Eldor R. Y col. (2004). treatment of diabetic foot ulcers: a comprehensive review of emerging treatment strategies Diabet Med.21 (11): 1161-73).

Methods have been developed to create skin substitutes that are placed over the ulcerative lesion. Dermagraft is produced from human dermis and fibroflasts on a synthetic structure of absorbable material and has been shown to be effective in low-grade ulcers with a higher rate of healing in a shorter time (Marston WA, et al. (2003) Dermagraft Diabetic Foot Ulcer Study Group. efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial (Diabetes Care 26: 1701-5). Apligraf consists of a dermal cap composed of human fibroblasts in a bovine type I decollagen matrix and an epidermis cap formed from human keratinocytes. Similarly, this skin substitute has been shown to be significantly associated with greater and faster wound healing when low-grade, uninfected neuropathic ulcers are applied (Veves A., et al (2001) Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized clinical trial (Diabetes Care 24: 290-5).

In a randomized, double-blind, placebo-controlled phase III clinical trial, the gel-shaped Platelet Derived Growth Factor (abbreviated PDGF) was shown to be effective and safe for the treatment of diabetic patients with neuropathic ulcers. good blood perfusion (Wieman TJ., et al (1998) Clinical efficacy of beclapermin (rh PDGF-BB) gel. DiabetesCare 21 (5): 822-7). Most patients (95%) included in this study had ulcers with an area <10 cm2 according to the evaluation by planimetry. Becaplermin 100 μg / g gel, compared with placebo, significantly increased the proportion of complete closure of the lesion by 43% (50 vs. 35%, ρ = 0.007) and reduced the time to achieve this effect by 32% (86 vs 127 days, ρ = 0.013). Satisfactory results with PDGF or becaplermin (Regranex) have led to its approval for treatment of neuropathic ulcers in the lower extremities of the diabetic that extend to subcutaneous or deeper tissue and have adequate blood flow (Brem H., Sheehan P., Boulton AJ. (2004) Protocol for treatment of diabetic foot ulcers, Am. J. Surg. 187 (5A): IS-IOS).

A method of administering a healing agent such as Epidermal Growth Factor (ECF) has recently been published, which consists of infiltrating a solution of the biomolecule into the lesion through multiple injections (WO 03/053458). This treatment has been shown to be effective in preventing diabetic foot amputation but has the drawback of traumatic patient outcome as injections of the lesion are very painful. Each injection should be given several injections and the patient should receive the treatment every other day for several weeks. Given the drawbacks of this method, the use of a sustained release formulation of FCE could reduce the frequency of drug administration which would be of considerable benefit to the patient.

There is a patent (US 6,086,863) in which growth regulating factors (eg Epidermal Growth Factor) may be included in prophylactic or therapeutic compositions of depolystyrene microspheres or other non-degradable polymers to improve wound repair process such as ulcers. diabetic foot by locally applying a suspension of these microspheres to an appropriate vehicle. Topical application has the limitation of having less control of the dose that reaches the place of action, since there are several factors that can interfere with absorption, including: the presence of necrotic tissue and local exudate, blood flow compromise, presence of enzymes. degrading FCE.

Therefore, an important problem in the treatment of diabetic doping foot ulcers is to achieve the effective dose of a drug that can regenerate ischemic tissue and prevent amputation of the diabetic foot.

Many other patents have focused on other methods of accelerating the level of healing. However, none of these methods will be widely effective.

Explanation of the invention

Generally, the present invention comprises a pharmaceutical composition containing Epidermal Growth Factor-loaded microspheres to be administered parenterally in the lower limbs of diabetic patients afflicted with chronic ischemic injuries to prevent amputation of said limbs. Related to this invention, the term microspheres include microspheres and nanospheres. Encapsulation in FCE microspheres allows: (i) controlled release of the drug and (ii) protection against degradation processes such as digestion with existing proteins in place of action.

In this invention, the microspheres can be described as polymeric matrix spheres with the active principle homogeneously dispersed throughout their volume, which is released in a controlled manner.

In the context of this invention, the term "controlled release" includes continuous, discontinuous, linear or nonlinear drug release. This is accompanied by the use of different polymer matrix compositions, inclusion of excipients that modify release profiles and / or degradation promoting agents or other modifications, which performed individually or in combination produce the desired effect on the properties of the composition.

The microspheres are obtained by the double emulsion / solvent evaporation method described by Okada et al. (US 4,652,441).

Preferred polymers for the development of this invention are those which, due to their properties, result in biocompatible and biodegradable properties. This latter condition is of paramount importance as it allows parenteral application of the formulation by infiltration of the lesion. Especially preferred are homopolymers of glycolic acid and lactic acid and the copolymer family derived from them (poly (lactide-co-glycolide), abbreviated PLGA). These polymers have characteristics that have made them excellent biomaterials for the manufacture of sutures, orthopedic fixation elements, and components of modified drug delivery systems (Ashammakhi N., et al (2001). Developments in Craniomaxillofacial Surgery: Use of Self-Reinforced Bioabsorbable Devices. Reconstruction Surgical Speeial Topie: 167-80 Eppley B. L. (2005) Use of resorbable plates and screws in pediatric facial fractures J. Oral Maxillofae Surg 63 (3): 385-91). It is among its properties that are biocompatible and biodegradable; In addition, the possibility of varying the release profiles of the active principle is due to the polymer matrix composition, the weight of the polymer and the addition of other excipients to the particles.

In addition to the family of lactic and glycolic acid polymers, other polymers with similar biocompatibility and biodegradability properties may be employed. They include polycaprolactone, hydroxybutyrate and hydroxyvalerate copolymers, lactic acid and caprolactone copolymers, polyorthoesters and polyanhydrides.

In a preferred embodiment, the microspheres of the pharmaceutical composition have a diameter in the range 1 to 100 μιη and the FCE constitutes 1.6-2.4% of the total mass of the microspheres.

In another preferred embodiment, the microsphere encapsulated FCE is released at a rate from the first day of infiltration in amounts of 5 to 10 μg per day and retains its physicochemical and biological properties for 14 days.

Another aspect of the present invention relates to the treatment of ischemic deletions of a diabetic patient by administering by local infiltration into the tissue comprising the lesion edges and bottom of the aforementioned pharmaceutical composition.

The microencapsulation of active principles of a protein nature pays particular attention to the activity of said biomolecules after the microencapsulation process. This is due to the fact that the proteins are mostly sensitive to the high temperatures that are often generated in the encapsulation processes and in the organic solvents employed to dissolve the polymers. On the other hand, each protein exhibits its own behavior regarding the processes of microencapsulation. Taking into account these aspects, the establishment of a methodology for the elaboration of conserved biological activity comprotein loaded microspheres requires a detailed study while the selection of the method to be employed, polymers, solvents, additives, etc.

As an active agent, the pharmaceutical composition may contain naturally obtained CEF by chemical synthesis or via recombinant DNA.

The pharmaceutical composition may also contain as a carrier a drug of the following groups: antimicrobials (penicillins, cephalosporins, quinolones, metronidazole, clindamycin, vancomycin, macrolides, tretracyclines, aztreonam and imipenem), anesthetics, non-steroidal analgesics, medicines angiogenic reaction (vascular endothelial growth factor, fibroblast growth factor), other growth factors (granulocyte colony stimulating factor) or erythropoietin.

The condition treated with the pharmaceutical composition of this invention is chronic ischemic skin lesion in the lower limbs of diabetic patients. Depending on the conditions of the injury and the characteristics of the patient being treated, it may be necessary for the microspheres present in the pharmaceutical composition to contain more than one encapsulated molecule. These additional therapeutic agents belong to the group of antimicrobials, anesthetics, painkillers from the non-steroidal anti-inflammatory group, drugs. with angiogenic action, other growth factors.

The pharmaceutical composition should be administered to the resuspended patients in a suitable vehicle, which may be in a first embodiment, a saline solution with viscosity agents such as carboxymethylcellulose, hydroxypropyl methylcellulose and detergents such as polysorbates or in a second embodiment a PEG-PLGA-PEG type hydrogensensitive. or derived from chitosan or dodextran.

The pharmaceutical composition object of this invention reduced the frequency of administration during treatment and unexpectedly increased the therapeutic benefit by reducing the total treatment time since wound healing was faster compared to that obtained by employing equivalent amounts of microencapsular FCE. The superior therapeutic effect of our formulation was not expected because the release lentoprofile it produces produces low concentrations of FCE. Unexpectedly also, another formulation in which excipients were used to release FCE faster and thus achieve higher drug concentrations did not have the therapeutic effects of the formulation of this invention.

Brief description of the figures.

Figure 1. Scheme representing the location of induced ulcers in the animal model.

Figure 2. Diagram of obtaining the FCE-loaded microspheres by the double emulsion - solvent evaporation method.

Figure 3. Microphotograph of an FCE-loaded microsphere.

Figure 4. Release profile of encapsulated FCE in PLGA microspheres. The χ axis shows the time in days and the y axis the amount of FCE released expressed as a percentage of the total amount contained in the microspheres employed in the assay, (♦) rapid release formulation and (·) slow release formulation.

Figure 5. Reverse phase chromatography analysis of trypsin digested FCE under different conditions. A: Control, B: Free FCE, C: FCE encapsulated in PLGA microparticles, D: FCE mixed with empty PLGA microparticles.

Detailed description of embodiments / Examples.

To give a more complete description of the invention the following examples are described. Example 1. Obtaining the pharmaceutical composition of encapsulated FCβ in PLGA microspheres.

Preparation of FCE-loaded microspheres.

A 50:50 or 10% (w / v) glycolic acid lactic acid copolymer solution (Sigma, St. Louis, Missouri, USA) was prepared by dissolving 1 g of the polymer in dichloromethane (DCM). In a glass container 1 ml of the polymeric solution was deposited and 200 µl of an aqueous 20 mg / ml FCE solution was added. This mixture was sonicated for 30 seconds using an IKASONIC U 200 Scontrol probe ultrasound (IKA Labortechnik, Germany). The resulting emulsion was poured into 40 ml of 1% polyvinyl alcohol and the second emulsion (w / o / w) was obtained by stirring the two phases at 14,000 rpm in an Ultraturrax T8 (IKALabortechnik, Germany). The double emulsion was poured into 140 ml of 30,000-70,000 polyvinyl alcohol (Sigma, St. Louis, Missouri, USA) at 0.1% and stirred in a homogenizer (IKA Labortechnik, Germany) at 300 rpm for 1h to evaporate dichloromethane. Finally, the microspheres were collected by filtration, washed 5 times with 50 ml of distilled water each time and freeze-dried in a lyophilizer (Edwards, UK). The dried microspheres were stored at 4 ° C until such time as they were used (Figure 2).

FCE microspheres with excipients were obtained similarly to that described but with the addition of Pluronic F-127 (10 mg) and NaCl (0.5mg) in the internal aqueous phase.

Characterization of FCE microspheres.

The process encapsulation efficiency and particle load were calculated by determining the concentration of CEF by the microBCA assay in the solution resulting from the digestion of the particles with IN NaOH which was neutralized with HCl IN prior to determination. As a result of the microencapsulation process they were obtained. spherical microparticles with regular surface and pore presence (Figure 3). Said encapsulated FCE microspheres were obtained with an average yield of 85%. 40-60% of the protein initially introduced in the manufacturing process was incorporated into the microspheres. The particles had a charge between 1.6 and 2.4%. The size of the microparticles was less than 25 μιη.

Table 1. Characteristics of FCE-loaded microspheres.

<table> table see original document page 11 </column> </row> <table>

The use of excipients did not significantly vary the characteristics of the obtained FCE-loaded microspheres.

In vitro release of encapsulated FCE.

Fifty mg of FCE-loaded microspheres were resuspended in 1 ml receptor fluid (0.001% Tween 80 and 0.1% sodium azide in PBS pH 7.2). The suspension was incubated at 37 ° C with moderate shaking. The samples at certain time intervals (0.25 (6h), 0.5 (12h), 1, 3, 7, and 14 days) were centrifuged for 5 min at 5000 rpm in a Hettich microliter centrifuge (Tuttlingen, Germany). The supernatant was collected and the same volume of fresh receptor fluid was added. The concentration of FCE in each extracted sample was determined by microBCA assay.

The release profile of the FCE contained in the microspheres is composed of a quick release step, which occurred during the first day and another step in which the release occurs continuously until 14 days. During the first stage approximately 20% of the total encapsulated protein was released for both preparations, while in the remaining period up to 65% of the FCE present in the excipient particles (in an approximate ratio of 28 μg per day) and up to 30 was released. % in the particles without excipients (at a rate of approximately 7 per day) (Figure 4).

Characterization of in vitro released FCE.

This experiment aims to demonstrate that encapsulated FCE conserves its physicochemical and biological properties. The properties of FCE released during the evaluation period (14 days) were studied.

FCE released during the first day, up to 7 and 14 days was characterized by the use of various analytical techniques: reverse phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gelplane electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay ( ELISA) biological activity. The results appear in table 2.

Table 2. Physicochemical and biological characteristics of the released FCE invitro.

<table> table see original document page 12 </column> </row> <table>

the percentage of immunoidentified FCE with respect to the aqual released mass was determined by the microBCA assay. b Percentage corresponding to the majority band detected at 6000 Da

c Percentage corresponding to principal speciesd FCE employed to obtain microspheres

The results show that the released FCE has similar physicochemical and biological characteristics to the employed FCE to obtain the microspheres.

Effect of microencapsulation on FCE stability against protease action.

1 mg FCE were separately prepared under three different conditions: (i) dissolved in 1 ml 4% sodium hydrogen carbonate (NaHCO3), (U) encapsulated in PLGA microspheres (2 wt%) and resuspended in 1 ml of 4% NaHCO3 and (iii) mixed with 50 mg of empty and resuspended PLGA microspheres in 1 ml 4% NaHCO3. Each preparation was added 100 μΐ of a 200 μg / ml trypsin solution in 4% NaHCO3 and allowed to incubate at 37 ° C for 4 hours with moderate shaking. As a control, one milligram of FCE in 1.1 ml of 4% NaHCO3 was used. After this time the reaction was stopped with 10 μΐ of trifluoracetic acid. Samples containing microspheres were centrifuged for 10 minutes at 6000 g and the supernatant separated from the precipitate. Microencapsulated or adsorbed FCE in the microspheres was separated from the polymer by the dichloromethane / acetic acid extraction method (Ruiz JM, et al (1989). Microencapsulation peptide: a study of the phase separation of poly (D, L-lactic acid-glycolyc acid ) copolymers 50/50 by silicone oil (J. Pham. Sci. 49: 69-77). All fractions were analyzed by RP-HPLC according to the procedure described by Han et al. (Han K., et al. (1998) Site-specific degradation and transport of recombinant human epidemic growth factor (rhEGF) in the mucous gastrointestinal rat. J. Pharm 168: 189-197). The results (Figure 5) show that both unencapsulated FCE and mixed with empty microspheres were completely degraded. The microencapsulated OCE, in turn, was protected from proteolysis to obtain a chromatographic profile similar to that of the control.

Example 2. In vivo effect (in an animal model) of encapsulated FCE vs free FCE.

Model of acute controlled experimental lesions.

The following experiment was conducted with the objective of evaluating the healing effect of the new infiltration or parenteral drug use, to be injected into the deferred edges and bottoms, based on microspheres containing CSF in satisfactory acute prognostic lesions.

Experimental biomodel: male Wistar rats of body weight between 225-250 grams. The animals were kept in controlled areas of the CIGB Biotério and under a stable lighting regime of 12 χ12 hours, air exchange cycles, as well as free access to diet. Individual rats were housed in T3 boxes with bed replacement every 48 hours with prior sterilization.

Ulcer induction: the animals were anesthetized with ketamine / xylazine acombination intraperitoneally. Mechanical and chemical backbone was performed from the backscapular space to the height of the sacrum. The region underwent asepsis with a solution of iodine-povidone and isopropyl alcohol. The skin site for inducing ulcers has been delimited with nanjing to induce circular full-thickness lesions with 9mm diameter disposable biotomes (AcuDrem, Fl, USA).

As shown in Figure 1, 6 equidistant symmetrical lesions were created in each animal. Once created, they were washed with sterile saline solution and their inner edge was delineated with indelible ink to calculate the wound area in zero time. Injuries from all animals in each group were daily sanitized with sterile 70% ethanol and saline to apply treatment if appropriate.

Experimental Groups:

After the ulcers were created, the animals were randomly numbered using a cross-order entry / group table to the following experimental groups:

Group I - it's not about anything. It is a spontaneous evolution control.Group II - placebo (given by the microspheres formulation vehicle: 0.3% carboxymethylcellulose solution, 0.1% Tween 20 and 0.9% locally infiltrated sodium chloride).

Group III - treatment by infiltration of the microspheres solution (without excipients), containing 675 μ§ of FCE, in one milliliter of the vein for this formulation. The infiltrations were performed on the deep edges of the wound.

Group IV - treatment by infiltration of the microspheres solution (with excipients), containing 675 FCE, in one milliliter of the vehicle for this formulation. The infiltrations were performed on the deep edges of the wound.

Group V - simple formulation of FCE in 0.9% saline physiological solution containing 75 μg FCE / ml.

10 rats were included for each group, so that 60 wounds per group were studied. Infiltration treatments were performed daily on animals treated with preparations that did not contain microspheres by inserting the needle (271/2) into the deferred edges and bottom, with prior sedation with diazepam intraperitoneally of the animals. Animals treated with the microencapsulated FCE formulations and the bead carrier were infiltrated only once.

Determination of wound closure level. Histological Processing:

Lesions were inscribed on transparent acetate slides for kinetic calculation of contraction at the following times: Time 0 - represents 100% of the open lesion area and 0% contraction, Time 1 - 72 hours induced, Time 2 - 5th day Induced, Time 3 - 7th day of induced, Time 4 - 9th day of induced. This day is taken as the end of the study and for the sacrifice of the animals according to the previous experience of spontaneous healing of these lesions. The images of the edges of the lesions were digitized. The area and percentage of contraction were calculated using the DIGIPAT image analysis program. Statistical calculations of each parameter were performed using the SPSS package using the nonparametric Mann Whitney U test, establishing a significance level of ρ <0.05.

Animals were sacrificed by sodium depentobarbital overdose (250 mg / kg) intraperitoneally. Lesions were resected from the fleshy panicle and fixed in 10% neutral formalin for later inclusion in paraffin. Hematoxylin / eosin, van Giesson and Masson's trichrome stains were used. The number of animals in each group with 100% epithelialization of the lesion, with differentiated and stratified epidermis was considered for each group.

The results of wound contraction kinetics are shown in table 3 (contraction values in mm expressed as a percentage over the calculation of maximum dilation at Time 0).

Table 3. Ulcer contraction values

<table> table see original document page 16 </column> </row> <table>

(*) Means statistical difference of ρ <0.05 with the rest of the groups. Proof of Man-Whitney U.

Unexpectedly the formulation containing FCE microspheres with slower release profile (without excipients) exerted the most potent wound edge contraction effects which in other words means that it exerts the most favorable effect on full healing acceleration. The contraction represents the convergence of several solid events that bring the wound closer to the remodeling phase.

Table 4 shows the percentage of the territory occupied by mature and organized granulation tissue of the ulcers in an experimental group. The calculations were performed on the samples collected at Time 4 by quantifying the number of positive microscopic fields coincidentally with van Giesson and Masson's trichrome reactions in the sample. Valuations were performed by two pathologists independently and blindly.

Table 4. Percentage of granulated territory at time 4 in each experimental group. Study of 60 wounds per group employing positive reactions to collagenous fibers.

<table> table see original document page 17 </column> </row> <table>

Unexpectedly the formulation of slower release profile microspheres of FCE (without excipients) exerted the most potent effects on the granulation tissue establishment and maturation process, which is as described for the wound contraction process explained above.

The effect of the treatments was also studied on the process of epithelialization of the lesions. The microscopic aspect of the epithelium was considered considering the total reepithelization of the ulcer, the presence of a stratified epithelium, and the existence of a keratin stratum. For the microscopic study the lesions were submitted to a longitudinalcentral hemisection and included in the same paraffin block. A total of 120 histological sections per group were studied, which essentially represent 60 lesions. The results are expressed in table 5.

Table 5. Effect of treatment on the epithelialization process. <table> table see original document page 18 </column> </row> <table>

* means p <0.05. Mann Whitney U test.

Group III, treated with the formulation of slow profile FCE microspheres (without excipients), surprisingly shows the best indicators of epithelial response, data for total epithelialization and epithelial maturity.

Chronic Skin Ulcer Model.

The following experiment was conducted to evaluate the healing effect of the new infiltration-based pharmaceutical formulation based on microspheres containing FCE in a model of chronic prognostic lesions that mimic those frequent in diabetic patients.

Experimental biomodel: Male Wistar rats weighing 225-250 grams. The animals were kept in controlled areas of the CIGB Biotério and under a stable lighting regime of 12 χ12 hours, air exchange cycles, as well as free access to diet. Individual rats were housed in T3 boxes with bed replacement every 48 hours with prior sterilization. The animals had been previously treated for two months with a 0.01% methylglyoxal solution to create a glycosylation environment comparable to that in a long-term diabetic patient. Among other organic damages is the enrichment of the deferred granulation process and remodeling (Berlanga J., et ao (2005) Methylglyoxal administration inducesdiabetes-like microvascular changes and disorders of the healing process of cutaneous wounds. Clin. Sci. (Lond) 109 (l) : 83-95)

Ulcer induction: Animals were anesthetized with ketamine / xylazine acombination intraperitoneally. Mechanical and chemical backbone was performed from the backscapular space to the height of the sacrum. The region underwent asepsis with a solution of iodine-povidone and isopropyl alcohol. The skin site for inducing ulcers has been delimited with nanjing to induce circular full-thickness lesions with 9mm diameter disposable biotomes (AcuDrem, Fl, USA). Six symmetrical and equidistant lesions were created in each animal, such as was done for the previous study. Once created, they were washed with sterile saline solution and their inner edge was delineated with indelible ink to calculate the wound area in zero time. Lesions from all animals in each group were daily sanitized with 70% ethanol and sterile saline before applying treatment in the corresponding case. Experimental groups:

After the ulcers were created, the animals were randomly numbered using an entry / treatment cross-table to the following experimental groups:

Group I - it's not about anything. It is a spontaneous evolution control.

Group II - placebo, given by the microspheres formulation vehicle: 0.3% carboxymethylcellulose solution, 0.1% Tween 20 and 0.9% locally infiltrated sodium chloride.

Group III - treatment by infiltration of the microspheres solution (without excipients), containing 1 mg FCE, in one milliliter of the vein for this formulation. The infiltrations took place over the edges and bottom of the wound.

Group IV - treatment by infiltrating the microspheres solution (with excipients), containing 1 mg FCE, in one milliliter of the vein for this formulation. The infiltrations were performed on the wound edges and bottom.Group V - simple formulation of FCE in 0.9% saline physiological solution, containing 75 μg of FCE / ml.

10 rats were included for each of the groups, so that 60 wounds per group were studied. Treatments were performed daily in animals treated with preparations that did not contain microspheres, with previous sedation with diazepam intraperitoneally of the animals. Animals treated with the microencapsulated FCE formulation and the bead carrier were infiltrated only once.

Determination of wound closure level. Histological Processing:

Lesions were inscribed on transparent acetate slides for kinetic calculation of contraction at the following times: Time 0 - represents 100% of the open lesion area and 0% contraction, Time 1 - at 72 hours induced, Time 2 - on the 5th day. induced, Time 3 - on the 7th day of induced, Time 4 - on the 9th day of induced, Time 5 - on the 14 decreated days. This day is taken as the end of the study and for the sacrifice of the animals according to the previous experience of spontaneous healing kinetics of these lesions. The images of the edges of the lesions were digitized. The contraction area and percentage were calculated using the DIGIPAT image analysis program. Statistical calculations of each parameter were performed using the SPSS package using the nonparametric test of Whit Whit U, with a significance level of ρ <0.05.

Animals were sacrificed by sodium depentobarbital overdose (250 mg / kg) intraperitoneally. Lesions were resected from the fleshy panicle and fixed in 10% neutral formalin for later inclusion in paraffin. Hematoxylin / eosin, van Giesson and Masson's trichrome stains were used. The number of animals in each group with 100% epithelialization of the lesion, with differentiated and stratified epidermis was considered for each group. Wound contraction kinetic results appear in table 6.20

Table 6. Contraction of wounds during assessment time

<table> table see original document page 21 </column> </row> <table>

(*) Means statistical difference of ρ <0.05 with the rest of the groups.

(**) Means statistical difference of ρ <0.01 with the rest of the groups. Provade Mann-Whitney U.

Shrinkage values in mm are expressed as a percentage over the calculation of maximum dilation at Time 0.

Unexpectedly the formulation of FCE microspheres with the slowest release profile exerted the most potent wound edge contraction effects, which in other words means that it has the most favorable effect on accelerating total healing, whereas contraction represents the convergence of wounds. several consolidated events that bring the wound closer to the remodeling phase. It is noted that these wounds simulate the biochemical microenvironment of the diabetic wound in which the contraction mechanism is partially pathologically completely abolished.

Table 7 shows the percentage of territory occupied by mature and organized granulation tissue of chronic ulcers in each experimental group. Calculations were performed on the samples collected at Time 5, quantifying the number of positive microscopic fields coincident with the reactions of van Giesson and Mason's Trichrome in each sample. Valuations were performed by several pathologists and one consultant independently and blindly.Table 7. Percentage of grainy territory at Time 5 in an experimental group. Study of 60 wounds per group employing positive reactions to collagen fibers.

<table> table see original document page 22 </column> </row> <table>

** It means difference of ρ <0.01 with the rest of the groups. U MannWhitney test.

Unexpectedly, treatment with the slower release profile FCE microsphere-based formulation exerted the most potent effects on the granulation tissue establishment and maturation process, which corresponds to that described for the previously described wound contraction process.

The effect of the treatments was also studied on the process of epithelialization of the lesions. The microscopic aspect of the epithelium was considered considering the total reepithelization of the ulcer, the presence of a stratified epithelium, and the existence of a keratin stratum. For the microscopic study the lesions were submitted to a longitudinalcentral hemisection and included in the same paraffin block. A total of 120 histological sections per group were studied, which essentially represent 60 lesions. There was no need to eliminate any of the bacterial contamination lesions. Results are expressed in table 8.Table 8. Effect of treatment on epithelialization process.

<table> table see original document page 23 </column> </row> <table>

Group III, treated with the formulation of slow-release FCE microspheres, unexpectedly showed the best indicators of epithelial response, given by total reepithelization and epithelium maturity.

Example 3. In vivo effect (in patients with advanced pediabetic ulcers) of encapsulated CSF vs free CSF.

The formulation with slower-releasing FCE microspheres (without excipients) was administered to patients with pediabetic ulcers and increased amputation risk. A 58-year-old diabetic female patient with an ulcerative lesion of the right foot over an area of 30.5 cm2 and evidence of ischemic impairment of the affected limb was treated with the formulation object of the present invention. After performing the lesion debridement, the formulation was administered with slower-release CFF microspheres once every 15 days for one month by infiltrating the edges and bottom of the ulcerative lesion. Rapid formation of useful granulation tissue was observed since the first week of treatment initiation, reaching in the third week 100% coverage of the affected area. The patient evolved satisfactorily with complete closure of the lesion and avoiding the need to indicate amputation. The product was well tolerated and no adverse events were reported.

The perilesional and intralesional administration of this formulation favored the formation of granulation tissue and the closure of the lesion, which unexpectedly shortened the healing process with respect to previous treatments and prevented the need for amputation. This treatment modality has resulted in better tolerance given to the reduction in the number of administrations.

Claims (10)

1. Pharmaceutical composition containing epidermal growth factor microspheres characterized by the fact that it is administered parenterally in the chronic ischemic cutaneous lesions of the lower limbs of diabetic patients and to prevent amputation of said limbs. Pharmaceutical composition according to Claim 1, characterized in that the epidermal growth factor is encapsulated in polymeric microspheres or nanospheres. Pharmaceutical composition according to Claim 2, characterized in that the microspheres are made of a polymeric material from the group consisting of lactic acid and glycolic acid copolymers, lactic acid homopolymers, glycolic acid homopolymers, polycaprolactone, hydroxybutyrate copolymers and hydroxyvalerate, lactic acid and caprolactone copolymers, polyorthoesters and polyanhydrides. Pharmaceutical composition according to claim 2, characterized in that said microspheres have a diameter in the range of 10000 μιη. Pharmaceutical composition according to claim 2, characterized in that the epidermal growth factor constitutes 1.6-2.4% of the total mass of the microspheres. Pharmaceutical composition according to Claim 1, characterized in that the release rate of the epidermal decay factor from the first day is between 5 and 10 μg / day. Pharmaceutical composition according to claim 1, characterized in that the epidermal growth factor released during 14 days retains its physicochemical and biological properties. Pharmaceutical composition according to Claims 1 to 7, characterized in that it is used in the treatment of ischemic lesions of a diabetic patient. Pharmaceutical composition according to Claims 1 to 7, characterized in that it is administered by local infiltration into the tissue comprising the edges and bottom of the lesion. Pharmaceutical composition according to any one of claims 1 to 7, characterized in that it is resuspended in a PEG-PLGA-PEG type hydrogelsensitive or a chitosan or dodextran derivative.